Dissociation of Per1 and Bmal1 circadian rhythms in PNAS PLUS the suprachiasmatic nucleus in parallel with behavioral outputs Daisuke Onoa,1,2, Sato Honmab,1,3, Yoshihiro Nakajimac, Shigeru Kurodad, Ryosuke Enokia,b,e, and Ken-ichi Honmab aPhotonic Bioimaging Section, Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan; bDepartment of Chronomedicine, Hokkaido University Graduate School of Medicine, Sapporo, 060-8638, Japan; cHealth Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan; dResearch Institute for Electronic Science, Hokkaido University, Sapporo, 001-0020, Japan; and ePrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japan Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved March 28, 2017 (received for review August 11, 2016) The temporal order of physiology and behavior in mammals is The expression of Per genes in the SCN is activated by a timed primarily regulated by the circadian pacemaker located in the exposure to light, which phase shifts the circadian pacemaker (8, hypothalamic suprachiasmatic nucleus (SCN). Taking advantage of 9). The phase-dependent phase shifts of clock gene expression bioluminescence reporters, we monitored the circadian rhythms of are regarded as a key mechanism by which the circadian pace- the expression of clock genes Per1 and Bmal1 in the SCN of freely maker is entrained to a LD cycle. Light signals from the retina moving mice and found that the rate of phase shifts induced by a stimulate the expression of Per genes, perturbing the core loop single light pulse was different in the two rhythms. The Per1-luc dynamics to produce a phase-dependent phase shift (8). How- rhythm was phase-delayed instantaneously by the light presented ever, the mechanisms by which light-induced phase-shift signals at the subjective evening in parallel with the activity onset of be- from the core loop are transduced to circadian rhythms in phys- havioral rhythm, whereas the Bmal1-ELuc rhythm was phase- iology and behavior are not well understood. delayed gradually, similar to the activity offset. The dissociation On the behavioral level, the onset and offset of an activity was confirmed in cultured SCN slices of mice carrying both Per1-luc band (activity onset and offset) of circadian behavioral rhythm and Bmal1-ELuc reporters. The two rhythms in a single SCN slice are known to respond differentially to a phase-shifted LD cycle showed significantly different periods in a long-term (3 wk) cul- (10) and to a single light pulse under continuous darkness (DD) ture and were internally desynchronized. Regional specificity in in nocturnal rodents (11). In addition, the phase relation between the SCN was not detected for the period of Per1-luc and Bmal1- the activity onset and offset is known to change under different ELuc rhythms. Furthermore, neither is synchronized with circadian photoperiods (12). Furthermore, the two phases of behavioral + intracellular Ca2 rhythms monitored by a calcium indicator, rhythm occasionally split under the constant light condition (12). GCaMP6s, or with firing rhythms monitored on a multielectrode From these findings, the two oscillator hypothesis was advanced to array dish, although the coupling between the circadian firing and explain behavioral circadian rhythm in nocturnal rodents (13). Ca2+ rhythms persisted during culture. These findings indicate that the expressions of two key clock genes, Per1 and Bmal1, in the SCN Significance are regulated in such a way that they may adopt different phases and free-running periods relative to each other and are respec- The circadian clock in the suprachiasmatic nucleus (SCN) regu- tively associated with the expression of activity onset and offset. lates seasonality in physiology and behavior, which is best characterized by the change in the activity time of behavioral clock gene | in vivo recording | suprachiasmatic nucleus | rhythms. In nocturnal rodents, the activity time was shortened photic phase resetting | E and M oscillators in long summer days and lengthened in short winter days be- cause of the change in the phase relationship of activity onset n mammals, the circadian pacemaker in the hypothalamic and offset, for which different circadian oscillators are predicted. Isuprachiasmatic nucleus (SCN) entrains to a light–dark (LD) Taking advantage of in vivo monitoring of clock gene expres- cycle and regulates circadian rhythms of behavior and physiology sion in freely moving mice, we demonstrated that the circadian (1, 2). The circadian oscillation in the SCN is autonomous, and rhythms of Per1 and Bmal1 in the SCN are associated differen- the clock genes Per1, Per2, Cry1, Cry2, Clock, and Bmal1 play tially with the phase shifts of activity onset and offset, respec- crucial roles (3). A heterodimer of Clock and Bmal1 proteins tively, suggesting the existence of two oscillations with different (CLOCK/BMAL1) activates the transcription of Per and Cry molecular mechanisms in timing of circadian behavior. genes; in turn, the protein products of these genes suppress their own transactivation by CLOCK/BMAL1, closing a feedback Author contributions: D.O., S.H., and K.H. designed research; D.O. performed research; loop. One turn of the auto-feedback loop (core loop) takes D.O., S.H., Y.N., S.K., and R.E. contributed new reagents/analytic tools; D.O. and Y.N. ∼ constructed a method of simultaneous measurement of bioluminescence; D.O., S.H., and 24 h. On the other hand, Bmal1 expression is enhanced by R.E. constructed a method of fluorescence imaging; S.K. made an analytical program for RAR-related orphan nuclear receptor (ROR) and is repressed imaging; D.O., S.H., and K.H. analyzed data; and D.O., S.H., and K.H. wrote the paper. by an orphan nuclear receptor in the RevErb family (α, β) The authors declare no conflict of interest. through ROR response element (4, 5). The expressions of ROR This article is a PNAS Direct Submission. and the RevErb family are enhanced in turn by a BMAL1/CLOCK 1To whom correspondence may be addressed. Email: [email protected] or heterodimer via an upstream E-box. Thus, the Bmal1 circadian [email protected]. rhythm is auto-regulated by a feedback loop (the Bmal1 loop) 2Present address: Department of Neuroscience II, Research Institute of Environmental which is interlocked with the core loop, maintaining an antiphasic Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. phase relationship with Per1 rhythm. This interlocked Bmal1 loop 3Present address: Research and Education Center for Brain Science, Hokkaido University, has been considered to contribute to stabilization and fine tuning Sapporo 060-8638, Japan. of the core loop (4, 6) in addition to the regulation of downstream This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NEUROSCIENCE pathways (7). 1073/pnas.1613374114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1613374114 PNAS | Published online April 17, 2017 | E3699–E3708 Downloaded by guest on September 25, 2021 Local time A 0 12 0 12 0 Per1-luc (SCN) Behavior (counts/min) 120 80 0 90 60 60 40 7 Days 30 20 RLU (counts/min) 0 0 1 2 3 45678 91011121314 15 14 Days B Local time 0 12 0 12 0 Bmal1-ELuc (SCN) Behavior (counts/min) 80 0 120 90 60 60 40 7 Days 30 20 RLU (counts/min) 0 0 1 2 3 45678 91011121314 15 14 Days Light pulse C Local time Local time 6012 18 612186012 18 61218 1 1 7 7 Days Days Per1-luc Bmal1-ELuc 14 14 D Days after light pulse Days after light pulse Days after light pulse -1 0 123456789 -1 0 123456789 -1 0 1234 567 89 0 0 0 Per1-luc Per1-luc Bmal1-ELuc -2 Bmal1-ELuc -2 -2 -4 -4 -4 * * * Phase shifts(h) Phase shifts (h) -6 Phase shifts (h) -6 -6 Activity onset Activity offset Fig. 1. Light pulse-induced phase-delay shifts of circadian rhythms in the SCN and behavioral rhythms in freely moving adult mice. (A and B) Typical examples of phase response at CT11.5 are illustrated for the Per1-luc (A) and Bmal1-ELuc (B) rhythms in the SCN with a behavioral rhythm (black histogram) (Left)in double-plotting, in which the colored area indicates bioluminescence larger than the minimum value of a series, and in sequential plotting (Right), in which broken lines indicate raw data and solid lines indicate 4-h moving-averaged values (Per1-luc,blue;Bmal1-ELuc, green). The number of behavioral activities in 1-min intervals is indicated by black vertical bars. A yellow vertical bar indicates the time of the light pulse. (C) Mean acrophases ± SEM (horizontal bar) are illustrated for Per1-luc (blue circles) (Left) and Bmal1-ELuc (green circles) (Right) together with the mean activity onsets (gray triangles) and offsets (black squares). A yellow horizontal bar indicates the time of a light pulse. A horizontal gray and black bar at the top of each panel indicates the LD cycle to which mice had been entrained. (D) Daily phase shifts are reported by mean ± SEM (n = 4) for Per1-luc (blue circles) and Bmal1-ELuc (green circles) (Left) rhythms, for Per1-luc and two phase markers (onset and offset) of behavioral rhythm (Center), and for Bmal1-ELuc and the two phase markers (Right). The abscissa indicates the number of days after a light pulse. An asterisk indicates statistically significant difference (P < 0.05, two-way ANOVA with a post hoc t test) between Per1-luc and Bmal1-ELuc (Left), between Per1-luc and activity offset (Center), and between Bmal1-ELuc and activity onset (Right).